Highly purified gonadotropin compositions and process for preparing them

ABSTRACT

Purification process of humanPurification process of human urinary gonadotropins of high biological activity and chemical purity absolutely free of foreign contaminating materials derived from the use of biological reagents or chromatography dyes, from crude of gonadotropins. The high biological activity and chemically pure composition of human gonadotropins obtained by this process, are used for the treatment of infertility and are selected from the group of follitropin or menotropins, having a bioactivity greater than 2500 IU/mg protein as tested by biological assay in rats, for both FSH and LH hormones for menotropins and greater than 5000 IU/mg protein for follitropin having an FSH:LH ratio about 75:1. Pharmaceutical preparations of said gonadotropins free of these contaminating materials are also comprised within the present invention.

The present invention relates to gonadotropin compositions, particularlyto FSH (follicle stimulating hormone: follitropin) and menotropincompositions of high biological activity and to a method for preparingthese compositions from human urine crude of menopausal orpostmenopausal women. The chemical purity obtained is greater than 95%as measured by HPLC, whereas the specific biologic activity is greaterthan 2500 IU/mg protein for both FSH and LH hormones for menotropincomposition (Human Menopausal Gonadotropins; HMG) and greater than 5000IU/mg protein for FSH.

BACKGROUND OF THE INVENTION

The term “menotropins” is applied to a hormonal combination obtainedfrom menopausal and post-menopausal women's urine comprising twoglycoprotein hormones: follicle stimulating hormone and luteinizinghormone. These two hormones are secreted by the pituitary gland, andsubsequently metabolized and excreted in the urine.

Menotropins and follitropin have been long used in the therapeuticaltreatment of infertility disorders.

The role of FSH consists in acting on the ovarian follicles, promotingtheir rapid growth and maturity with subsequent ovulation or atresia.FSH along with LH is also involved in the biosynthesis of estradiol inthe ovarian follicles that it has stimulated. The internal theca is themain site of androgen follicular biosynthesis which is under the controlof LH. Controlled by FSH, this androgen is then aromatized in thegranulose cells and excreted to the blood torrent. Therefore, LH is anecessary constituent along with FSH in follicular stimulation.

Pituitary hormones FSH and LH. thyroid stimulating hormone (TSH) and theplacental hormone human chorionic gonadotropin (HCG) are closely relatedsince all of them are glycoproteins having in their structure twosubunits called α and β. Both subunits are bound by non-covalentinteraction. These subunits do not have any biological activityseparately. The α-chains of the four above-mentioned hormones arecommon, while the β-chains are different and provide a biologicalactivity characteristic of each hormone. However, important portions ofthe β-chain are also common, which can be particularly observed in LHand HCG β-chains.

Gonadotropins obtained from human urine have been partially purifieduntil reaching biosafety characteristics compatible with therequirements of an injectable pharmaceutical product, and have beencommercialized for more than 30 years in order to solve infertilityproblems; being in the form of an injectable grade pharmaceuticalpreparation, they are listed in the most important pharmacopoeias in theworld and have been approved for pharmaceutical use in practically allcountries all over the world.

The composition of the follitropin preparation can be described asfollows:

-   Follicle stimulating hormone: 75 or 150 International units of FSH-   Excipient (required for the lyophilization process, generally 5–20    mg of lactose) and other non-active proteins.

The composition of the menotropin preparation can be described asfollows:

-   Follicle stimulating hormone: 75 or 150 International units of FSH-   Luteinizing hormone 75 or 150 International units of LH-   Excipient (required for the lyophilization process, generally 5–20    mg of lactose) and other non-active proteins.

International units of FSH and LH are calculated by measuring thebiological activities in rats relative to an international standardprepared by the National Institute for Biological Standards and Control(NIBSC) dependent on the World Health Organization.

The requirement, imposed by Pharmacopoeias such as United StatesPharmacopoeia (USP XXIII) or British Pharmacopoeia (BP) in terms ofpurity of the starting material required for the preparation ofinjectable grade menotropins, consists in a starting material having anFSH activity greater than 40 IU/mg and an LH activity greater than 40IU/mg. In view of this, those starting materials heretofore used havebeen obtained by a manufacturing process ensuring an activity in therange of 70–150 IU FSH/LH per mg., which is more than enough to meet therequirements imposed by the health organizations as far as biosafety andeffectiveness are concerned.

However, the latest developments in terms of purification techniques andthe development of gonadotropins from recombinant origin have influencedthe need for preparations of highly purified native gonadotropins ofurinary origin, without the presence of impurifying proteins.

Some FSH and LH purification process have been described in the recentpast.

EP 0 322 438 B1 and the equivalent U.S. Pat. Nos. 5,128,453; 5,767,067and 5,840,857 refer to the preparation of follicle stimulating hormonewith high specific activity from urinary origin. However these processesinclude a specific monoclonal antibodies step. From the safety point ofview, this fact raises some concerns about contamination of the productwith heterologous proteins, DNA residues from the hosts cell andviruses. Although process validations and final testing can help toexclude the possibility of potential contamination, it is reasonable tojudge that a process that is specially designed to avoid usingbiological reagents has higher safety standards than the one whichincludes this type of purification step.

The WO 98/20039 patent application describes a FSH and LH separation andpurification process which avoids employing biological reagents.However, due to the very high FSH:LH ratio of the raw material used andproducts obtained, this process excludes the possibility of obtainingmenotropins, a preparation which requires an FSH:LH ratio ofapproximately 1:1

Moreover, WO 98/20039 fails to characterize the gonadotropins obtainedas a bioactive material, which is an essential issue for a therapeuticalproduct. This patent application uses an immunoradiometric method totest the activity, a determination method that arises deep concernsabout its accuracy.

In fact, it has been long recognized that the structural heterogeneityof FSH and LH isoforms has an important impact on the biologicalactivity and immunological reactivity of both hormones [Costagliola et.al. J. Endocrinol. Invest. 17, 291 (1994)]. Observed differences in bio-and immuno-FSH and LH levels suggest that separated structural entitiesare recognized by the bioassays versus the immunoassay. It has beenthoroughly discussed that immunoassays does not consistently provide agood estimation of the bioactive gonadotropins level and does notnecessarily reflect the biological activity [Dahl and Stone, J.Andrology, 13, 11 (1992); Rose et. al Endocrine reviews 21, 5 (2000)].

On the other hand, bioassays are considered to be the proper test todefine the hormone activity since it takes into account two importantcomponents that are absent in others methods: the biological action atthe target tissue and the biological clearance [Rose et. al. Endocrinereviews 21, 5 (2000)]. This fact makes the in vivo bioassay mandatoryfor calibration of therapeutics preparations [Rose, Clinica Chimica Acta273, 103 (1998)].

It must be also underlined that the WO 98/20039 fails also tocharacterize the gonadotropins as a chemically pure drug since noanalytical method is presented to support this fact.

The present invention presents a full characterization of thegonadotropins not only from the bioactivity scope but also from theanalytical point of view by introducing sensitive analytical method totest the purity. Since the assays used to establish the FSH and LHactivity in the present invention are, in all the cases, in vivobioassays which has been long proved to be a robust specific test forassuring bioactivity, the potency of the FSH and the LH for eachgonadotropins obtained and also their ratio can be assured.

A second issue to consider is that the above mentioned patent (WO98/20039) describes the use of an affinity dye chromatography to purifygonadotropins. This procedure could arise some concern about thepotential contamination of the product by the leakage of the dye. Thesekind of foreign compounds are not desirable in products to be injectedinto humans [Protein Purification by R. K. Scopes, Springler-Verlag NewYork Inc; 2nd Edition, page 156 (1998)]. Since the present invention wasdesign to exclude this type of chromatography, the dye contaminationproblem is an issue completely avoided.

Current menotropins (the commercially available product) may suffer thedrawback of potential local allergic reactions when they areadministered subcutaneously, due to the presence of proteincontaminants.

This invention provides a method for the purification and thepreparation of the first commercially available high purity gonadotropinproducts. This methodology was specially designed so as to avoid usingbiological reagents such as antibodies, receptros and other heterologusmaterials and chromatography dyes. Besides the safety issue, the use ofnon-specific steps during the isolation allows to obtain of all theisoforms that are present in the starting material.

As extensively described, heterogeneity is of particular importance inthe glycoprotein hormones. At least 20–30 different isoforms of FSH, LHand TSH exist (Wide, Acta Endocr., Copenh 109, 181–189, 1985). Theseisoforms differ in their molecular weight and charge. Althoughglycoprotein hormone isoforms mainly vary in the oligosaccharidestructure, microheterogeneities may be also present from differences inthe amino-acid composition (Costagliola et al, J. Endocrinol. Invest.,17, 291–299, 1994; Wilson et al., J. Endocrinol 125, 3–14, 1990).

It is generally accepted that the type of different isoforms isolateddepends, among other factors, on the isolation techniques (Cockburn etal., Biologicals 19, 257–264, 1991). The use of highly discriminatingtechniques for isolation, such as monoclonal antibodies, can contributeto select only one part of the isoforms present in the source of thematerial. Being “too specific”, some biologically active gonadotropinvariants may not be recognized by the antibodies and could be lost(Costagliola et al, J. Endocrinol. Invest., 17, 291–299, 1994). On theother hand, a non-selective approach, such as the one described in thispatent, can be useful in purifying all the types of isoforms present inthe urine. Different isoforms have shown to vary with respect to theinteraction with the cell surface receptors and metabolic clearance(Thotakura et al., Glycobiology 5, 3–10, 1995). Oligosaccharidestructure is under the control of various physiological factors. Fromthe therapeutical point of view, it is interesting to have a product inwhich all isoforms present in the urine are available. In this way, thehighly purified products described here provide the differential rolesof naturally occurring gonadotropin isoforms in the maintenance ofreproductive function.

It must also be underlined that the present application is the firstcharacterization of a highly purified menotropins preparation that wasconfirmed not only by its high biological potency but also by relevantscreening methods (PAGE electrophoresis, HPLC, size exclusionchromatography).

Being now available in a purified grade and in a large scale, thesenaturally occurring FSH and LH molecules can be then investigated andcarefully analyzed. The three dimensional structure of the hormones cancontribute to a better understanding about the role of theoligosaccharides.

The method for obtaining currently produced menotropins is well known.As indicated in BP 1980, menotropins can be prepared frompost-menopausal women's urine by kaolin adsorption, subsequent alkalielution and precipitation with 2 volumes of acetone. The requiredfraction is extracted from this precipitate with an ammonium acetatesolution in 70% ethanol and then precipitated with a 10% ammoniumacetate solution in 90% ethanol. The menotropins product is obtained byionic exchange chromatography of this precipitate.

The starting material, from now on the “HMG source material”, that canbe used in the present invention for obtaining highly purifiedmenotropins, constitutes the menotropins specialty, as specified in thepharmacopoeias (USP XXIII, BP 1993 addendum 1995; EP 1986) or any othermaterial closely related to this specialty. Therefore it is understoodthat this preparation process is also applicable to other materials thatdo not strictly meet the requirements applicable to menotropins. Infact, satisfactory results were obtained using Fraction C (seedescription of the invention. Example 1 and example 2), which is amaterial that falls to comply with the FSH:LH ratio menotropinsrequirement (FSH:LH, 1:1, approximately) Moreover, the present inventionprovides a procedure to adjust the FSH:LH ratio when needed beingtherefore capable of providing highly purified menotropins specialtyeven when starting materials which are out of the correct FSH:LH ratioare used. Thanks to this, it is possible to obtain a product comprisedof FSH and LH in the appropriate ratio 1:1 necessary to produce thepharmaceutical preparation, without having separated both activeprinciples during purification. That is to say, accomplishing theco-purification of both hormones, from a purity degree of approximately5% in the starting material to more than 95% in the final product with afinal potency 25–35 times greater than the initial one. The purificationdegree as obtained can be visualized from FIG. 1, which shows the resultof polyacrylamide gel electrophoresis of the conventional pharmaceuticalproduct vs. the new purified version obtained from the application ofthe present Invention.

The steps described in the present invention can be used following in adifferent order from the one herein described, which does not implyvarying the invention philosophy. Equally satisfactory results wereobtained by inserting, for example, the hydrophobic interaction resinbetween the two ionic exchange chromatography steps described later.

This invention also provides for the preparation of urinarygonadotropins separately, that is to say FSH and LH.

The administration of menotropins compositions for therapeutic purposeshas been carried out, mainly by intramuscular injection. Thisadministration form creates a considerable discomfort in the patient andrequires from the patient regular visits to clinical units, sometimesfor weeks or months in order to receive the treatment.

Subcutaneous administration would make the self-administration possibleand consequently improve patient's cooperation and compliance.

The subcutaneous administration of urinary gonadotropins has alreadybeen described (Nakamura Y., et al, Fertil Steril 51, 423–429, 1989;Engmann L. et al. Fertil Steril. 69, 836–840, 1998). The subcutaneousadministration of non-pure preparations may suffer the drawback of localallergies due to the presence of impurities in the product used andconsequently result in the suspension of the treatment. Therefore it isworthwhile to produce high purity products which can diminish thepossibility of these allergic reactions.

SUMMARY OF THE INVENTION

The present invention relates to FSH and/or LH compositions of highbiological activity and a method for preparing these compositions fromhuman urine crude of menopausal or postmenopausal women. The chemicalpurity is greater than 95% as measured by HPLC, whereas the specificbiologic activity is greater than 2500 IU/mg protein for the FSH and LHfor menotropins and greater than 5000 IU/mg for follitropin.

The compositions of the invention may comprise: any conventionalexcipient such as hexoses, mannitol and mixtures thereof, a stabilizerselected from the group of albumin, detergents and mixtures thereof.

The injectable compositions obtained are substantially free ofcontaminants and can be adapted for subcutaneous administration.

The follitropin and/or menotropins compositions of the present inventionmay be used to prepare a pharmaceutical preparation of high biologicalactivity and chemical purity that comprises or not a pharmaceuticallyacceptable carrier.

The separation and purification process of human urinary gonadotropinsof high biological activity and chemical purity from crude ofgonadotropins, including HMG source material, absolutely free of foreigncontaminating materials derivated from the use of biological reagents orchromatography dyes comprising mainly the following steps, in any order:

-   -   1) purification of said gonadotropins crude diluted in 0.05–0.15        M ammonium acetate, pH 5.0–6.0, in an ionic exchange column with        a strong cationic resin of the type of sulphopropyl, eluting        both FSH and LH with solutions of 0.05–0.5 M ammonium acetate,        pH 5.0–7.0; and    -   2) purification of this fraction diluted in 0.01–0.05 M ammonium        acetate, pH 5.0–7.0 in a column of ionic exchange with a strong        anionic resin of the ammonium quaternarium type, eluting both        FSH and LH with solutions of 0.05–0.2 M ammonium acetate, pH        5.0–7.0; and    -   3) purification of gonadotropins in a column with an hydrophobic        interaction resin by the sequencial addition of at least two of        the following solutions:    -   a) buffer of 50–200 mM sodium phosphate, and 0.8–1.2 M ammonium        sulfate, pH 5.0–6.0;    -   b) buffer of 50–200 mM sodium phosphate, and 0.4–0.6 M ammonium        sulfate; pH 5.0–7.0;    -   c) buffer of 50–200 mM sodium phosphate (50–70% v/v), and        ethanol 96% (50–30% v/v).        wherein said steps may be performed in any order.

Resins of the type of SP-Sepharose, Q-Sepharose and hydrophobicinteraction resins may be used in these steps. A preferred hydrophobicinteraction resin is a Phenyl-Sepharose resin.

The process of the invention further comprises secondary steps ofprecipitation, centrifugation, ultrafiltration, dialysis, washing,drying in vacuo, and cooling. The last step of this process may beselectively performed to obtain fractions that have only menotropins,FSH or LH activities.

One of the main advantages of the process of the present invention isthat the process does not include biological reagents and substrates andthat the product is free of impurifying biomaterials.

Starting material used in the present invention for producing highlypurified menotropins and highly purified FSH is essentially obtained bya very well known method which employs kaolin to adsorb gonadotropinsfrom the menopausal/postmenopausal urine (BP 1980). Briefly, bioactivefraction is extracted from the acidified urine with kaolin, eluted withalkali and precipitated with 2 volumes of acetone. Then, the bioactivefraction is extracted from this precipitate with 10% w/v solution ofammonium acetate in ethanol (70%) and precipitated with 10% ammoniumacetate in ethanol (90%). Further purification is done by ion-exchangechromatography. The purified material obtained by this processconstitutes the menotropins composition or some other equivalentpreparation, like Fraction C.

Menotropins or equivalent (Fraction C) is chromatographed on a strongcationic exchange column, with the active fraction being eluted with a0.2–0.5 M ammonium acetate buffer, pH 5.0–7.0. A new precipitate isobtained (Fraction F) by adding 4 volumes of ethanol. Fraction F ischromatographed on a strong anionic exchange column, with the activefraction being eluted with a 0.05–0.2 M ammonium acetate buffer, pH5.0–7.0. The active fraction is frozen at −75° C. (Fraction G).

Fraction G is analyzed by a biological assay to determine precisely itsFSH and LH content. Depending on the ratio found between both hormones,the following chromatographic step is taken, with two alternatives beingpossible:

1) co-purification of both hormones.

2) separation of the hormone activity which is in excess so as to obtaina Menotropins with 1:1 FSH/LH. Alternatively, separation of both FSH andLH provides both activities separately for therapeutic purposes (neatFSH and neat LH).

Once the steps to be taken are determined, Fraction G is dialyzed andconcentrated so as to adapt it to the needs of the followingchromatographic step. The resulting solution is chromatographed on ahydrophobic interaction column, eluting the active fractions accordingto two alternative processes taking into account options 1) and 2)above. The liquid fractions that are obtained (Fraction J2, J3, J4) arefrozen at −75° C. Afterwards, these fractions are defrozen, dialyzed andconcentrated, filtered through a sterilizing membrane and precipitatedby adding 4 volumes of ethanol, obtaining a precipitate that is dried invacuo to dryness (Fraction K).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents an electrophoretic run of the conventional product(HMG) vs. the new highly purified menotropins product (Fraction KM).

FIG. 2 represents a PAGE (Polyacrylamide Gel Electrophoresis) run ofhighly purified Menotropins (Fraction K_(M)) run in differentconcentrations together with the molecular weight standards.

FIG. 3 represents a PAGE run of a highly purified FSH (Fraction K_(F))in different concentrations together with the molecular weightstandards.

FIG. 4 represents a PAGE run of: 1) Gonal-F (Serono) 1 FSH IU/μl, 2)Metrodine HP (Serono) 3 FSH IU/μl, 3) Highly Purified Menotropins(Fraction K_(M)) 3.7 FSH IU/μl and 4) Molecular Weight Standards (fromthe top) 14,400 D, 20,100 D, 30,000 D, 43,000 D, 67,000 D and 94,000 D.

FIG. 5 represents an Isoelectric focusing on 3–10 PhastGels silverstained, wherein: Lane 1 corresponds to the Calibration Kits for Broad3–10 pI., Lane 2 corresponds to pI 4.55 standard (soybean trypsininhibitor), Lane 3 corresponds to pI 5.85 standard (bovine carbonicanhydrase B), and Lanes 4, 5 and 6 correspond to highly purifiedfollitropin (K_(F)) product (3 different production batches).

FIG. 6 represents an Isoelectric focusing on 3–10 PhastGels silverstained, wherein Lane 1 corresponds to the Calibration Kits for Broad3–10 pI. and Lane 2 corresponds to highly purified menotropins (K_(M))product.

FIG. 7 is the result obtained by HPLC with a Highly Purified Menotropins(Fraction K_(M)) sample.

FIG. 8 is the result obtained by HPLC with a Highly Purified FSH(Fraction K_(F)) sample.

FIG. 9 is the result obtained by HPLC with a sample of Gonal-F (Serono).(The peak at retention time 11.2 corresponds with an excipient of thepharmaceutical preparation), and

FIG. 10 is a process Flow chart for obtaining the highly purifiedgonadotropins of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

a) Flow chart

The flow chart for obtaining highly purified gonadotropins is shown inFIG. 10.

The elaboration technique of Fraction K_(M) constituting purifiedMenotropins will be described below.

b) Preparation of Fraction F

Fraction C is chromatographed on a chromatographic column containing 10liters of strong cationic exchange resin of the sulphopropyl type.

Fraction C (110–140 g) is dissolved in 1600–1800 ml of a 0.05–015 Mammonium acetate solution, pH 5.0–7.0. The column is run and eluted withthe necessary amount of 0.05–0.15 M ammonium acetate solution to bringthe volume to 20 liters. The elution is continued with solutions of0.15–0.20 M ammonium acetate, pH 5.0–7.0 (20 liters) and 0.2–0.5 Mammonium acetate, pH 5.0–7.0 (20 liters). The active fraction elutedwith the latter solution is added with stirring to 4 volumes of 96%ethanol and enough acetic acid to reach a mixture pH of 5.5–5.7. Aprecipitate is formed, separated by centrifugation, washed with ethanoland dried in vacuo until ethanol is removed and humidity is lower than5% (Fraction F).

c) Preparation of Fraction G

Fraction F is chromatographed on a chromatographic column containing 4liters of strong anionic exchange resin of ammonium quaternary type

Fraction F (40–60 g in 650 ml) is dissolved in 0.01–0.05 M ammoniumacetate solution, pH 5.0–7.0, the column is run and eluted with the samesolution to bring the volume to 7 liters. Elution is continued with 12liters of 0.05–0.07 M ammonium acetate pH 5.0–7.0, then with 10 litersof 0.07–0.2 M ammonium acetate pH 5.0–7.0. The active fraction elutedwith the latter solution is subjected to an ultrafiltration processusing a PM 10 (10000 D) Ultrafilters (Amicon-Millipore) membrane. Thesolution is concentrated and dialyzed against 50 mM sodium phosphatebuffer, pH 5.5–5.7 to a concentration of 2–4 g of protein in 100–150 mlof buffer. Then it is frozen at −75° C. (Fraction G).

d) Preparation of Fraction J

Fraction G is chromatographed on a chromatographic column containing 400ml of a hydrophobic interaction resin (Phenyl Sepharose HP,Amersham-Pharmacia Biotech).

A sufficient amount of ammonium sulfate is added to the solution ofFraction G to obtain a 0.8–1.2 M concentration.

The chromatographic process to be carried out will allow theco-purification of FSH and LH or the separation of both hormones. Thecourse of action will depend on the prior analyses conducted withFraction G (biological assays), through which the FSH:LH ratio has beendetermined. Once this ratio is known, the overstock of the hormone inexcess of 1:1 FSH:LH ratio will be removed.

d-1) If the product is balanced (1:1 FSH:LH), the chromatography ofconcentrated and dialyzed Fraction G will be conducted as follows:

Put the solution of Fraction G in the chromatographic column, 0.8–1.2 Min ammonium sulfate.

Elute with 2 volumes of 50–200 mM sodium phosphate buffer, 0.8–1.2 Mammonium sulfate, pH 5.0–7.0.

Continue the elution with 2 volumes of 50–200 mM phosphate buffer (50–70% v/v) and 96% ethanol (50–30% v/v).

The active fraction eluted with the latter buffer (Fraction J4) isfrozen at −75° C. This fraction has FSH and LH activity.

d-2) If the FSH:LH ratio of Fraction G is different from 1:1, theoverstock of the hormone in excess will be removed as follows:

Run an aliquot of the solution of fraction G in the chromatographiccolumn. 0.8–1.2 M in ammonium sulfate.

Elute with 2 volumes of 50–200 mM sodium phosphate buffer, 0.8–1.2 Mammonium sulfate. pH 5.0–7.0.

Continue the elution with 2 volumes of 50–200 mM sodium phosphatebuffer, 0.4–0.6 M ammonium sulfate, pH 5.0–7.0, and finally with 2volumes of 50–200 mM phosphate buffer (50–70% v/v) and 96% ethanol(50–30% v/v).

The active fraction eluted with 50–200 mM sodium phosphate buffer,0.4–0.6 M ammonium sulfate (Fraction J2) is frozen at −75° C. Thisfraction has mostly FSH activity.

The active fraction eluted with 50–200 mM phosphate buffer (50–70% v/v)and 96% ethanol (50–30% v/v) (Fraction J3) is frozen at −75° C. Thisfraction only has LH activity.

Preparation of Fraction K

Fractions J2, J3, J4 are defrozen, dialyzed and concentrated usingultrafiltration through a PM 10 (Diaflo Ultrafilters, Amicon-Millipore)membrane against a 50 mM sodium phosphate buffer, pH 5.7.

Each resulting solution is filtered using a 0.45μ membrane under thenecessary conditions to obtain a sterile product, and then added to 4volumes of 96% ethanol and enough acetic acid to obtain a mixture pH of5.5–5.7. The mixture is allowed to stand overnight. The precipitate isseparated by centrifugation and dried in vacuo until ethanol is removedand humidity is lower than 5% (Fraction K).

Properties of Fraction K may vary depending on the precipitated fractionbeing J2, J3 or J4. Fraction K_(M) obtained from Fraction J4 willcontain approximately equivalent units of FSH and LH. Fraction K_(F)obtained from Fraction J2 will be comprised of FSH and LH traces.Fraction K_(L) obtained from Fraction J3 will be comprised of LH and FSHtraces.

EXAMPLES OF BATCHES PRODUCED BY THE DESCRIBED TECHNIQUES Example 1

1.1 Preparation of Fraction F

231.2 g of Fraction C were divided in two equal portions andchromatographed in two equivalent processes on a chromatographic columnas above described.

115.6 g of Fraction C (in each process) were dissolved in 1700 ml of0.05 M ammonium acetate buffer, pH 5.0 The column was run and elutedwith further 18.7 liters of the same chromatographic buffer. The elutionwas continued with 20 liters of 0.15 M ammonium acetate buffer, pH 5.0and finally with 20 liters of 0.5 M ammonium acetate buffer, pH 5.0 Theactive fraction obtained by eluting with 0.5 M ammonium acetate (22liters) was added with stirring to a solution of 88 liters of 96%ethanol and 2400 ml of acetic acid. The pH of the mixture was 5.7. Theprecipitate obtained was left in the refrigerator (2–8° C.) overnight.The precipitate was centrifuged, washed with 96% ethanol and dried invacuo for 17 h.

On each of the two equivalent processes, two Fractions F of 20.7 g and19.5 g were obtained, respectively.

1.2 Preparation of Fraction G

The two fractions F obtained in the above step were brought together andchromatographed on column according to the described technique.

40.2 g of Fraction F were dissolved in 650 ml of 0.01 M ammonium acetatebuffer, pH 5.0. The column was run with this solution and eluted with6350 ml of the same dilution buffer. The elution was then continued with12 liters of 0.05 M ammonium acetate, pH 5.0 and then with 10 liters of0.2 M ammonium acetate, pH 5.0. The active fraction (4500 ml) elutedwith the latter solution was subjected to an ultrafiltration processusing a PM 10 (10000 D) Diaflo Ultrafilters (Amicon-Millipore) membrane.The solution is concentrated and dialyzed against 50 mM sodiumphosphate, pH 5.7 for obtaining a concentration of 2–4 g of protein in150 ml of buffer. Final solution (400 ml) was frozen at −75° C.

Fraction G was biologically tested in animals detecting a FSH potency of42.000 IU/ml and LH potency of 33,780 IU/ml. With this result, it wasconsidered necessary processing a portion of the solution (80 ml) ofFraction G under conditions for separating FSH and LH fractions J2 andJ3 respectively). The rest (320 ml) was chromatographed under conditionsso as not to separate both hormones (fraction J4). Aliquots of FractionJ3 and Fraction J4 where then mixed to obtain a final FSH:LH ratio ofapproximately 1:1 (fraction K_(M)).

1.3 Preparation of Fraction J

i) Preparation of Fractions J2 (Highly Purified FSH) and J3 (HighlyPurified LH):

Ammonium sulfate was added to an aliquot of fraction g (80 ml) until aconcentration 1 M. This solution was run in Phenyl-Sepharose HPchromatographic column and was eluted with 2 volumes of buffer, 50 mMsodium phosphate, 1 M sulfate ammonium, pH 5.1. The elution wascontinued with 2 volumes of buffer, 50 mM sodium phosphate, 0.5 Mammonium sulfate, pH 5.1, and finally with 2 volumes of 50 mM sodiumphosphate buffer (60% v/v) and 96% ethanol (40% v/v).

The FSH active fraction eluted with buffer, 50 mM sodium phosphate, 0.5M ammonium sulfate, pH 5.1 (Fraction J2) was dialyzed and concentratedusing PM 10 membrane ultrafiltration (Diaflo Ultrafilters,Amicon-Millipore), against a 50 mM sodium phosphate buffer, pH 5.7, andthen was frozen at −75° C.

The LH active fraction eluted with 50 mM sodium phosphate buffer (60%v/v) and 96% ethanol (40% v/v) (Fraction J3) was dialyzed andconcentrated using PM 10 membrane ultrafiltration (Diaflo Ultrafilters,Amicon-Millipore), against a 50 mM sodium phosphate buffer, pH 5.7, andthen was frozen at −75° C.

ii) Preparation of Fraction J4 (Highly Purified Menotropins)

A second aliquot of Fraction G (320 ml) was defrozen and ammoniumsulfate was added until a 1 M concentration was obtained. This solutionwas run in a Phenyl-Sepharose HP chromatographic column and was elutedwith 2 volumes of buffer 50 mM sodium phosphate, 1 M ammonium sulfate,pH 5.1. The elution was continued with 2 volumes of phosphate 50 mM (60%v/v) and 96% ethanol (40% v/v). The eluted fraction with this buffer(Fraction J4) was frozen at −75° C.

1.4 Preparation of Fraction K

Fractions J3 (40 ml) and J4 (25 ml) were defrozed, filtered through0.45μ membrane under necessary conditions for obtaining a sterileproduct (final volume 100 ml), and then admixed and stirred with 4volumes of 96% ethanol (400 ml) and acetic acid necessary for reaching apH 5.5 (1 ml).

Fraction J2 (40 ml) was defrozen, filtered through a 0.45μ membraneunder necessary conditions for obtaining a sterile product (final volume60 ml), and then admixed and stirred with 4 volumes of 96% ethanol (400ml) and acetic acid necessary for reaching a pH 5.5 (0.5 ml).

Fractions were allowed to precipitate in the refrigerator overnight at2–8° C. The next morning, the highly purified menotropins precipitate,obtained from Fraction J3 y J4 was separated by centrifugation and driedin vacuo until ethanol was removed and moisture was lower than 5%(Fraction K_(M), 4.50 g).

The highly purified FSH precipitate obtained from the J2 fraction wasseparated by centrifugation and dried in vacuo until ethanol was removedand humidity was lower than 5% (Fraction K_(F), 0.55 g).

1.5 Results

The biological analysis performed with Fractions K_(M) (highly purifiedmenotropins) and K_(F) (highly purified FSH) exhibited the followingresults:

FSH Potency Specific Activity LH Potency Fraction (IU/mg) (IU/proteinmg) (IU/mg) K_(M) 2,870 3,700 2,635 K_(F) 6,895 8,900 <1 LH IU/75 FSH IU

Yield Table Yield relative Total Yield Prior stage FSH LH Fraction FSHIU LH IU FSH % LH % % % C 19,276,000 15,604,000 — — — — F 18,408,00014,980,000 95.5 96.0 95.5 96.0 G 16,800,000 13,512,000 91.3 90.2 87.286.6 K_(M) 12,915,000 11,857,000 76.9 87.8 67.0 76.0 K_(F)  3,792,000   50,600 22.6 — 19.7 —

Example 2

2.1 Preparation of Fraction F

250.06 g of Fraction C were divided in two equal portions andchromatographed in two equivalent processes in a chromatographic columnas the one described above.

125.03 g of Fraction C (in each process) were dissolved in 1,700 ml of0.05 M ammonium acetate buffer, pH 5.1. Then, the column was run andeluted with further 18.7 liters of the same chromatographic buffer. Theelation was continued with 20 liters 0.15 M ammonium acetate buffer, pH5.1, and finally with 20 liters of 0.5 M ammonium acetate buffer, pH5.1. The active fraction obtained by elution with 0.5 M ammonium acetate(22 liters) was added under stirring to 88 liters of 96% ethanol and2,200 ml of acetic acid. The mixture pH was 5.7. It was observed theappearance of a precipitate. The mixture was left in the refrigerator at2–8° C. overnight. The precipitated was centrifuged, washed with 96%ethanol and dried in vacuo for 22 hs.

In each of the equivalent processes, two fractions F of 21.57 g and21.15 g were respectively obtained.

2.2 Preparation of Fraction G

The two fractions obtained in the previous stage were brought togetherand chromatographed on column according to the process described above.

42.58 g of Fraction F were dissolved in 650 ml of 0.01 M ammoniumacetate buffer, pH 5.1. This solution was run in the column and elutedwith 6.350 ml of the same buffer dissolution. The elution was continuedwith 12 liters of ammonium acetate 0.05 M, pH 5.0, and then with 10liters of ammonium acetate 0.2 M, pH 5. The active fraction (4,500 ml)eluted with this last solution was subjected to an ultrafiltrationprocess with a PM 10 membrane (10,000 D) (Diaflo Ultrafilters,Amicon-Millipore). The solution was concentrated and dialyzed against a50 mM sodium phosphate buffer, pH 5.7, until a concentration of 2–4 g ofprotein in 150 ml of buffer is obtained. The final volume of 500 ml wasfrozen at −75° C.

Fraction G was biologically tested in animals, showing a FSH potency of49,790 IU/ml and a LH potency of 39,600 IU/ml. With this analysis, itwas considered necessary processing one part of the solution (100 ml) ofFraction G under conditions for separating FSH and LH (fractions J2 andJ3 respectively) and the rest (400 ml) under conditions so as not toseparate both hormones (J4).

Aliquots of Fraction J3 and Fraction J4 where then mixed to obtain afinal FSH:LH ratio of approximately 1:1 (Fraction K_(M)).

2.3 Preparation of Fraction J

i) Preparation of Fraction J2 (Highly Purified FSH) and J3 (HighlyPurified LH):

Ammonium sulfate was added to one aliquot of Fraction G (100 ml) until a1 M concentration is obtained. This solution was run in thePhenyl-Sepharose HP chromatographic column and was eluted with 2 volumesof buffer 50 mM sodium phosphate, 1 M ammonium sulfate, pH 5.1. Then theelution is continued with 2 volumes of buffer 50 mM sodium phosphate,0.5 M ammonium sulfate, pH 5.1, and finally with 2 volumes of 50 mMsodium phosphate (60% v/v) and 96% ethanol (40% v/v).

The FSH eluted active fraction with buffer 50 mM sodium phosphate. 0.5 Mammonium sulfate, pH 5.1 (Fraction J2) was dialyzed, concentrated usingmembrane ultrafiltration with PM 10 membrane (Diaflo UltrafiltersAmicon-Millipore), against a 50 mM sodium phosphate buffer, pH 5.7, andthen was frozen at −75° C.

The LH eluted active fraction with 50 mM sodium phosphate buffer (60%v/v) and 96% ethanol (40% v/v) (Fraction J3) was dialyzed, concentratedusing membrane ultrafiltration with a PM 10 membrane (DiafloUltrafilters, Amicon-Millipore), against a 50 mM sodium phosphatebuffer, pH 5.7, and then was frozen at −75° C.

ii) Preparation of Fraction J4 (Highly Purified Menotropins):

To a second aliquot of Fraction G (400 ml) ammonium sulfate was addeduntil a 1 M concentration was obtained. The solution was run inPhenyl-Sepharose HP chromatographic column and was eluted with 2 volumesof buffer 50 mM sodium phosphate, 1 M ammonium sulfate, pH 5.1. Theelution was continued with 2 further volumes of 50 mM phosphate buffer(60% v/v) and 96% ethanol (40% v/v). The active fraction eluted withthis buffer (Fraction J4) was frozen at −75° C.

2.4 Preparation of Fraction K

Fractions J3 (50 ml) and J4 (30 ml) were defrozen, filtered through a0.45% membrane under necessary conditions for obtaining a sterileproduct (final volume 110 ml), and then were added under stirring to 4volumes of 96% ethanol (440 ml) and enough acetic acid to achieve a pH5.5 (1 ml).

Fraction J2 (50 ml) was defrozen, filtered through a 0.45μ membraneunder necessary conditions for obtaining a sterile product (final volume70 ml), and then was added under stirring to 4 volumes of 96% ethanol(280 ml) and enough acetic acid to achieve a pH 5.5 (0.5 ml).

Fractions were allowed to precipitate in the refrigerator overnight at2–8° C. The next morning, the highly purified menotropins precipitate,obtained from Fractions J3 and J4 was separated by centrifugation anddried in vacuo until ethanol was removed and moisture was lower than 5%(Fraction K_(M), 5.71 g).

The highly purified FSH precipitate, obtained from Fraction J2, wasseparated by centrifugation, dried in vacuo until ethanol was removedand moisture was lower than 5% (Fraction K_(F), 0.70 g).

2.5 Results

The biological analysis performed with Fractions K_(M) (highly purifiedmenotropins) and K_(F) (highly purified FSH) showed the followingresults:

FSH Potency Specific Activity LH Potency Fraction (IU/mg) (IU/proteinmg) (IU/mg) K_(M) 3,344 4,300 3,022 K_(F) 6,500 8,400 <1 LH IU/75 FSH IU

2.6 Yield Table Yield relative Total Yield prior stage FSH LH FractionFSH IU LH IU FSH % LH % % % C 29,000,000 21,900,000 — — — — F 27,800,00021,200,000 95.9 96.8 95.9 97.0 G 24,900,000 19,800,000 89.6 93.4 85.990.4 K_(M) 19,094,000 17,255,000 76.7 87.1 65.8 78.8 K_(F)  4,550,000    <60,600 18.3 — 15.7 —

High purified products were also obtained using the process of thepresent invention starting with less active materials. In this case anFSH of about 5000 IU/mg protein and menotropins of a potency of about2500 IU/mg protein for both FSH and LH were obtained.

2.7 Characterization of the Obtained Products

Fractions K_(A1) and K_(F) were characterized by the followingtechniques:

-   2.7.a) Polyacrylamide gel Electrophoresis (PAGE)-   2.7.b) Polyacrylamide gel Electrophoresis followed by Western-blot    analysis-   2.7.c) Isoeiectrofocusing-   2.7.d) Size exclusion chromatography (SEC) in HPLC-   2.7.e) Protein contents measurement-   2.7.f) Biological potency dosage in animals (previously informed)    2.7.a) Polyacrylamide gel Electrophoresis (PAGE)

Fractions K_(M) and K_(F) were analyzed by electrophoresis according tothe following procedure:

-   Equipment: Ultrathin Polyacrylamide Gel Electrophoresis System,    PhastSystem (Amersham Pharmacia Biotech).-   Gels: Phast Gel gradient 8–25 (Amersham Pharmacia Biotech).-   Buffer: Buffer strips/SDS (Amersham Pharmacia Biotech).-   Separation Technique: File 110, PhastSystem, SDS-Page-   Development Technique: File 200, PhastSystem for Coomasie Brilliant    Blue.-   Low Molecular Weight Probes: Electrophoretic Calibration Kit    containing 6 purified proteins (Amersham Pharmacia Biotech).

Molecular Weight Phosphorylase b 94,000 D Albumin 67,000 D Ovoalbumin43,000 D Carbonic Anhydrase 30,000 D Trypsine inhibitor 20,100 Dα-lactalbumin 14,400 D

Each kit vial contains a lyophilized blend with approximately 100 μg ofeach protein. Each vial was dissolved with 100 μL of sample buffer.

Sample Buffer:

250 mg of SDS and 0.5 ml of β-mercaptoethanol were dissolved in 10 ml ofbuffer A.

Buffer A: EDTA 1 mM 372 mg TRIS 10 mM 1.21 mg H20 q. s. t. 1,000 ml pH8.0

The samples were dissolved so that the final concentration was1,100–1.300 FSH IU/ml of sample buffer.

Sample treatment

The sample was heated at 100° C. for 5 minutes. Blue bromophenol wasadded until a 0.01% concentration was obtained.

After the Electrophoretic run and the development, the gels were driedwith hot air.

Obtained Results

The electrophoresis runs of Fractions K_(M) (FIG. 2) and K_(F) (FIG. 3)gave as result a profile in which it is observed in an almostexclusively way a unique band developed with Brilliant Coomassie Bluewith a migration distance midway of the standards of molecular weight20,100 D and 30,000 D, indicating an approximate molecular weight of25,000 D.

Sample Rf Std. 20,100 D 0.196 Fractions K_(M)–K_(F) 0.250 Std. 30,0000.300

The assignment of the observed band in the electrophoresis of FractionsK_(F) and K_(M) was performed by two ways:

a) by comparison with 2 commercial products containing FSH as the soleactive ingredient: Gonal-F (Serono) containing FSH of recombinantorigin, Metrodine HP (Serono) containing FSH of urinary origin (see FIG.4).

b) by Electrophoresis-Western blot as was indicated in 2.7.b).

2.7.b) Polyacrylamide Gel Electrophoresis followed by Western-Blotanalysis.

The samples of fractions K_(M) and K_(F) were analyzed by Western blot.After performing a polyacrylamide electrophoresis process in gradientsimilar to the one described in 2.7.a), the bands were transferred to anitrocelluose support and developed by antibody action. Specificantibodies for chain β-FSH, chain β-LH and against chain α of bothhormones were used.

Technique: The transfer technique No. 221 was used for the PhastSystem(Amersham Pharmacia Biotech), employing the following transfer buffer:

Transference buffer: Tris 25 mM, Glycine 192 mM, pH 8.3, containing, 20%methanol.

Membrane: Probind 45, 0.45 μm pore

Transfer conditions: 25 V, 25 mA, 1 W, 45 min.

Stained with Coomassie Blue:

Staining Solution: 0.1% solution of Phast Gel Blue R in methanol 30% andacetic acid 10% in distilled water.

Final Solution: Mix 1 part of the staining solution with 1 part ofacetic acid 20% in distilled water.

Procedure: Color the membrane in the final solution for 30 minutes withgentle stirring. Wash the membrane with solution ofmethanol:water:acetic acid (30:60:10) twice and then with acetic 20%.

Procedure of detection: The detection method by streptavidine-biotinewas used.

Buffer used: PBS (sodium phosphate 0.01 M, sodium chloride 0.25 M, pH7.6).

Blocking solution: Albumin 5% in PBS.

Washing solution: a) Albumin 5% in PBS, b) PBS.

Primary antibody solution: The following primary antibodies were used.

1) anti β-LH monoclonal antibody (Immunotech) (IgG1-mice), Catalogue No.0374.

2) anti β-FSH monoclonal antibody (Immunotech) (IgG1-mice), CatalogueNo. 0373.

3) monoclonal antibody against α-subunit of pituitary hormones(Immunotech) (IgG1-mice), Catalogue No. 0375.

Dilution: dilute the primary antibody 1:100.

Secondary antibody solution: The secondary antibody used was:Biotin-SP-conjugated AffiniPure F(ab′)₂ Fragment Goat Anti-Mouse IgG (H+L), (heavy chain and light chain). (Immunotech, Cat. No. 0816) diluted1:500 in PBS.

Peroxidase-conjugated Streptavidine solution: A dilution 1:500 in PBS ofPeroxidase-conjugated Streptavidine (Immunotech, Cat. No. 0309) wasused.

Development solution: Horseradish Peroxidase conjugate substrate kit(Bio Rad, Cat No. 170-6431), containing a solution blend of oxygenatedwater, 4-chloro-1-naphtol and buffer for developing the color, was usedfor preparing 1 liter of solution.

Brief Description of the Procedure:

1) incubate the membrane with blocking solution overnight.

2) wash with washing solution a) twice for 5 min. each time.

3) incubate with primary antibody solution overnight.

4) wash with washing solution a) three times for 5 minutes each time.

5) incubate with secondary antibody for 1 hour.

6) wash with washing solution a) three times for 5 minutes each time.

7) incubate with conjugated peroxidase for 30 minutes.

8) wash with washing solution a) three times for 5 minutes each time.

9) incubate with developing solution for 10 minutes.

10) stop the reaction.

Results

After performing the polyacrylamide gel electrophoresis of FractionsK_(M) and K_(F), the bands were transferred to nitrocellulose membranesaccording to the above informed technique, and developed. The followingresults were found.

Frac- tion Anti-β FSH antibody Anti-β LH antibody Anti-α-LH antibodyK_(M) Positive Positive Positive K_(F) Positive Negative Positive

In view of these results, it is concluded that the band developed withCoomassie Blue in the electrophoresis of fraction K_(M) had both FSH andLH activities. Instead, fraction K^(F) only reacted positively againstthe specific antibody for FSH, and not for LH. Given that α-chain of FSHand LH are common, both fractions KM and KF showed a positive reactionwith an antibody against the α-chain.

2.7.c) Isoelectrofocusing

Fractions K_(M) (highly purified FSH) and K_(F) (highly purified FSH)were analyzed by isoelectrofocusing according to the followingprocedure:

-   Equipment: Ultrathin Polyacrylamide Gel Electrophoresis System,    PhastSystem (Amersham Pharmacia Biotech).-   Gels: Phast Gel IEF 3–9 (Amersham Pharmacia Biotech).-   Separation Technique: File 100, PhastSystem.-   Development Technique: Silver Kit (Amersham Pharmacia Biotech).-   PI Standards: IEF calibration kit; Broad pI Kit 3–10) (Amersham    Pharmacia Biotech). Soybean trypsin inhibitor, pI 5.85(Sigma).    Bovine carbonic anhydrase, pI 4.55 (Sigma).    Sample Treatment

Samples were dissolve to have a concentration of 2.5 mg/ml to 1.25mg/ml.

After the electrophoretic run and the development, the gels were driedwith hot air.

Obtained Results

The pI distribution for both the K_(F) (highly purified FSH) and K_(M)(highly purified menotropins) are shown in FIG. 5 and FIG. 6. The acidicnature of the gonadotropins is confirm by the IEF pattern. In fact, asfully described in the literature, isoforms are restricted to the acidicrange.

2.7.d) Size exclusion Chromatography (SEC) in HPLC

Description of the equipment:

High performance liquid chromatograph Shimadzu, LC-10AVP, with manualinjector 7725i, with position sensor and loop of 20, 50 or 200 μL,Rheodyne.

UV-visible Spectrophotometer detector., model SPD-10AVP, Shimadzu(190–600 nm).

Working station for processing chromatographic data Shimadzu Class-CR10, program Class CR10 and module CBM-101.

Chromatographic column Bio-Rad BIO-SIL™ SEC250 silica-based sizeexclusion column (300×7.5 mm)

Flow: 1.0 ml/min.

Detection: UV at 220 nm.

Column Temperature: room temperature

Injection volume: 50 to 200 μL.

Sample preparation: Inject approximately 1 ml of the mobile phase in thevial containing the sample, stir until dissolution.

Results:

The following chromatograms of Fractions K_(M) and K_(F) showed thepresence of only one peak at a retention time of approximately 8.1–8.2sec. The retention time coincides with the one obtained bychromatography of a commercial product, Gonal-F (Serono) containingrecombinant FSH. (See FIGS. 7, 8 and 9)

2.7.e) Protein Dosage:

Method: The method of Lowry [Journal of Biological Chemistry 193, 265(1951)] with a Folin-Ciocalteu reactive, and a standard curve ofalbumin.

Results: The protein percentage for both fractions K_(M) and K_(F)indicated in examples 1 and 2 was approximately 77%.

2.7.f) Biological Potency Dosage in Animals.

As it was previously reported in section 2.5, fractions K_(M) and K_(F)were biologically analyzed in rats.

Methods:

2.7.f.1) FSH Biological Potency Dosage

The Steelman-Pohley method [Steelman, S. L. & Pohley, F. M.,Endocrinology 53, 604 (1953)] of ovarian weight increase was used inimmature 21–24 days-old female rats, injected with three doses of aproduct containing FSH. The doses should keep a ratio such that thedifference between the logarithms of the greater dose and the mediumdose is equal to the difference between the logarithms of the mediumdose and the smaller dose. Animal lots were used in which the weightdifference between the heaviest and the lightest animal was not morethan 10 grams. The animals were injected subcutaneously during threedays with three different doses of the sample dissolved inphosphate/albumin buffer and the corresponding doses of a standard. Onthe fifth day the animals were sacrificed, the ovaries were extractedand weighted. The data obtained with sample were compared with the dataobtained with the standard and the potency of the different samples wascalculated using the statistical scheme indicated for the analysis of asample against standard in a 3×3 test (see Biological Analysis of USPXXIII).

2.7.f.2) LH Biological Potency Dosage

The method of weight increase of seminal vesicle in immature 21–24days-old male rats injected with three doses of a product containing LHwas used. The three doses should keep a ratio such that the differencebetween the logarithms of the greater dose and the medium dose is equalto the difference between the logarithms of the medium dose and thesmaller dose. Animal lots were used in which the weight differencebetween the heaviest and the lightest animal was not more than 10 grams.The animals were injected subcutaneously during four days with threedifferent doses of the sample dissolved in phosphate/albumin buffer andthe corresponding doses of a standard. On the fifth day the animals weresacrificed, the seminal vesicles were extracted and weighted. The dataobtained with sample were compared with the data obtained with thestandard and the potency of the different samples was calculated usingthe statistical scheme indicated for the analysis of a sample againststandard in a 3×3 test (see Biological Analysis of USP XXIII).

The standard used was a sample of Menotropins calibrated against the 3rdinternational standard of urinary FSH and LH prepared by the NIBSC(National Institute of Biological Standards and Control—Great Britain)depending on the WHO (World Health Organization).

Example 3 Pharmaceutical Preparations

Highly purified injectable menotropins specialty:

Excipients that may be used in the composition are lactose, mannitol,and mixtures thereof. Other conventional excipients can also be used.

In the present invention, lactose was used as an excipient in theinjectable preparation.

The preparation pH can be corrected to a value in the range of 6,0–7,0by adding acids or bases (phosphoric acid or others and/or sodiumphosphate or others).

3 ml borosilicate glass type I vials with bromobutyl stoppers are usedas containers.

Preparation of a Batch of 5,000 Ampoules

The calculated amount of menotropins of high purity (with a 10%overfilling) is dissolved in 500 ml of water for injection. On the otherhand, 100 gr. of lactose is dissolved in 4 liters of water forinjection. Both solutions are mixed, the pH is adjusted, if necessary,by the addition of an acid or base, the resulting solution is completedto 5,000 ml and sterilized by filtration through a of 0.2μ membrane.Vials are filled with the prepared solution (1 ml) and loaded into asterile lyophilizer at a temperature of –40° C. for at least 8 hr. Thelyophilization starts heating at 3° C./hr up to temperature of +30° C.,which is maintained till the end of the cycle.

The present example is similarly applied for the preparation of highlypurified follitropin.

1. A purification process for obtaining the human urinary gonadotropinsfollicle-stimulating hormone (FSH) and/or luteinizing hormone (LH) ofhigh biological activity and chemical purity absolutely free of foreigncontaminating materials derived from the use of biological reagents orchromatography dyes, from crude gonadotropins comprising FSH LH, saidprocess comprising the following steps: 1) purifying said gonadotropinsdiluted in 0.05–0.15 M ammonium acetate, pH5.0–6.0, in an ionic exchangecolumn with a strong cationic sulfopropyl resin, eluting both FSH and LHwith solutions of 0.05–0.5 M ammonium acetate, pH 5.0–7.0; and 2)purifying the gonadotropins diluted in 0.01–0.05 M ammonium acetate, pH5.0–7.0, in an ion exchange column with a strong anionic resin of thequaternary ammonium type, eluting both FSH and LH with solutions of0.05–0.2 M ammonium acetate, pH 5.0–7.0; and 3) purifying thegonadotropins in a column with an hydrophobic interaction resin bysequential addition of at least two of the following solutions: a)buffer of 50–200 mM sodium phosphate, and 0.8–1.2 M ammonium sulfate, pH5.0–6.0; b) buffer of 50–200 mM sodium phosphate, and 0.4–0.6 M ammoniumsulfate; pH 5.0–7.0; c) buffer of 50–200 mM sodium phosphate (50–70%v/v), and ethanol 96% (50–30% v/v), and 4) recovering purified FSHand/or LH; wherein steps 1 to 3 may be performed in any order.
 2. Thepurification process of claim 1 wherein the strong cationic resin instep 1) is a SP-Sepharose resin.
 3. The purification process of claim 1wherein the strong anionic resin in step 2) is a Q-Sepharose resin. 4.The purification process of claim 1 wherein the hydrophobic interactionresin in step 3) is a Phenyl-Sepharose resin.
 5. The purificationprocess of claim 1, wherein the FSH is eluted with solution b, and theLH is eluted with solution c, in the addition sequence a, b, c in step8.
 6. The purification process of claim 1, wherein if the ratio of FSH:LH is approximately 1:1, the gonadotropins are eluted with solution c,in an orderly addition sequence a, c in step
 3. 7. The purificationprocess of claim 1, wherein if the ratio of FSH: LH is different from1:1, the process further comprises the addition of gonadotropin FSHeluted with solution b, or the addition of gonadotropin LH eluted withsolution c, in an orderly addition sequence a, b, c in step
 3. 8. Thepurification process of claim 1, wherein said crude gonadotropins areobtained from urine of menopausal and/or post-menopausal women.
 9. Thepurification process of claim 1, wherein said crude gonadotropins haveFSH activity greater than 40 IU/mg and LH activity greater than 40IU/mg.